This application claims priority from German Application No. DE 199 53 206.0, filed on Nov. 5, 1999, the subject matter of which is hereby incorporated herein by reference.
1. Field of the Invention
The present invention provides the novel plasmids pTET3 and pCRY4 and the use thereof for the production of vector plasmids.
2. Background Information
Naturally occurring plasmids and plasmid vectors produced therefrom are vital to the improvement of the production characteristics of coryneform bacteria. Constructing plasmid vectors for this group of industrially significant bacteria is substantially based on cryptic plasmids, which are provided with suitable selection markers capable of functioning in Corynebacteria or Brevibacteria (U.S. Pat. No. 5,158,891 and U.S. Pat. No. 4,500,640). These plasmid vectors may be used for cloning and amplifying genes which are involved in the production of L-amino acids, vitamins or nucleotides. Expression of these particular genes may have a positive influence on the production of the desired substances. Thus, for example, cloning a DNA fragment which encodes a protein for a lysine exporter resulted in an improvement in the fermentative production of L-lysine with Corynebacterium glutamicum strain MH20-22B (DE-A 19548222).
In contrast with the known and equally industrially significant bacterium Escherichia coli, only a limited number of natural plasmids and suitable selection markers are available for developing cloning and expression vectors for Corynebacteria and Brevibacteria. Many plasmids known by different names prove to be identical on more detailed analysis of their genetic organisation. These plasmid isolates have thus been classed in two groups (Sonnen et al., Gene 107, 69-74 (1991)).
The pBL1 group includes the plasmids pAM286 from Corynebacterium glutamicum AJ11560 (EP-A 0093611), pAM330 from Brevibacterium lactofermentum ATCC13869 (Miwa et al., Agricultural and Biological Chemistry 48, 2901-2903 (1984)), pX18 from Brevibacterium lactofermentum ATCC21086 (Yeh et al, Gene 47, 301-308 (1986)) and pBL1 from Brevibacterium lactofermentum ATCC2179B (Santamaria et al., Journal of General Microbiology 130, 2237-2246 (1984)).
The pHM1519 group comprises plasmids pCG1 from Corynebacterium glutamicum ATCC31808 (U.S. Pat. No. 4,617,267), pHM1519 from Corynebacterium glutamicum ATCC13058 (Miwa et al., Agricultural and Biological Chemistry 48, 2901-2903 (1984)), pSR1 from Corynebacterium glutamicum ATCC19223 (Yoshihama et al., Journal of Bacteriology 162, 591-597 (1985)) and pRN3.1 from Corynebacterium glutamicum ATCC39269 (U.S. Pat. No. 4,559,308).
In addition to members of these two groups of plasmids, the cryptic plasmids pCG2 from Corynebacterium glutamicum ATCC31832 (U.S. Pat. No. 4,489,160) and pAG3 from Corynebacterium melassecola 22220 (U.S. Pat. No. 5,158,891) have also been isolated.
The only selection systems which have hitherto been available are two antibiotic resistance markers which were identified on the streptomycin/spectinomycin resistance plasmid pCG4 from Corynebacterium glutamicum ATCC31830 (U.S. Pat. No. 4,489,160) and on the tetracycline resistance plasmid pAG1 from Corynebacterium melassecola 22243 (U.S. Pat. No. 5,158,891). Plasmid pCG4 also bears the sulI gene which imparts sulfamethoxazole resistance, the sequence of which gene was determined by Nesvera et al. (FEMS Microbiology Letters 169, 391-395 (1998)).
If strains which produce amino acids, vitamins or nucleotides are to be rapidly investigated and improved, it is important to have plasmid vectors which are mutually compatible and are sufficiently stable.
It is known from the prior art that pHM1519 plasmid derivatives and pEL1 plasmid derivatives may coexist. It is furthermore known that the plasmids pGA1 and pGA2 described in U.S. Pat. No. 5,175,108 are compatible. Plasmid vectors having high, moderate or low copy numbers so that expression of the gene under consideration may be graduated are also of significance. Most known plasmids have a high copy number. Only the plasmid pGA2 described in U.S. Pat. No. 5,175,108 is known to have a low copy number.
The widely used plasmid vectors are composed of components originating from the species C. glutamicum and components from another species of bacteria, typically E. coli. This method introduces foreign DNA into the species C. glutamicum. Functional plasmid vectors with a graduated copy number which contain only endogenous DNA and thus meet the criteria of self cloning are not known in specialist circles.
It is an object of the invention to provide novel plasmids that are suitable for constructing plasmid vectors having improved characteristics for coryneform bacteria which produce amino acids, vitamins and nucleotides.
Amino acids, vitamins and nucleotides are used in animal nutrition, in the food industry, in the pharmaceuticals industry and in human medicine. These substances are produced with strains of coryneform bacteria. Production characteristics are improved by amplifying suitable genes by means of plasmid vectors. There is accordingly general interest in providing novel plasmid vectors having improved characteristics.
The present invention provides the mutually compatible plasmids pTET3 and pCRY4, isolated from the strain of Corynebacterium glutamicum deposited under DSM number 5816, wherein
1.1 plasmid pTET3 is characterised by a length of xcx9c27.8 kbp and the restriction map shown in FIG. 1, and an antibiotic resistance region and
1.2 plasmid pCRY4 is characterised by a length of xcx9c48 kbp and the restriction map shown in FIG. 2.
The present invention also provides composite plasmids of pTET3 and pCRY4 capable of autonomous replication in coryneform bacteria, said plasmids containing
2.1 a part or the entire quantity of the nucleotide sequences
2.2 at least one DNA replication region derived from one of the plasmids pTET3 or pCRY4
2.3 optionally a gene fragment which is derived from a plasmid which can multiply in E. coli, B. subtilis or Streptomyces and
2.4 at least one region for expressing active substance resistance, preferably from the plasmid pTET3.
The present invention also provides novel composite plasmids that contain at least part of an active substance resistance region.
The novel plasmid pTET3, the restriction map of which is, shown in FIG. 1, has
1. a replication region comprising the nucleotide sequence shown in SEQ ID NO:1 and
2. an antibiotic resistance region consisting of a tetA gene imparting tetracycline resistance and an aadA gene imparting streptomycin and spectinomycin resistance, shown in SEQ ID NO:6.
The novel plasmid pCRY4, the restriction map of which is shown in FIG. 2, has a replication region comprising the nucleotide sequence shown in SEQ ID NO:4.
The present invention also provides the production of amino acids, vitamins and nucleotides using plasmid vectors (composite plasmids) which contain pTET3 and pCRY4 and optionally pGA1 or pGA2 nucleotide sequences.
Corynebacterium glutamicum LP-6, which was deposited as DSM5816 in the context of EP-B 0 472 869, contains the novel plasmids pTET3 and pCRY4 in addition to the plasmids pGA1 and pGA2 described therein. The storage period for DSM5816 has been extended pursuant to rule 9.1 of the Budapest Treaty.
Plasmids pTET3 and pCRY4 are prepared by culturing strain LP-6 in a conventional medium, such as for example brain-heart bouillon or Luria-Bertani medium. The cells were harvested by centrifugation, treated with lysozyme and digested by the alkaline lysis method. The DNA is then purified by anion exchange chromatography on silica gel particles, precipitated with ethanol or isopropanol and then resuspended in H2O. Complete systems for isolating plasmid DNA are commercially available as xe2x80x9ckitsxe2x80x9d. One example of such a kit is the xe2x80x9cNucleoBond Plasmid Kitxe2x80x9d from Clonetech Laboratories GmbH. The person skilled in the art will find detailed instructions relating to the use of this kit in the manual xe2x80x9cNucleoBond Nucleic Acid Purification Kits and Cartridges, User Manual (PT3167-1)xe2x80x9d from Clonetech Laboratories GmbH (Heidelberg, Germany, 1997). Plasmids pTET3 and pCRY4 are revealed as plasmid bands by separating the total plasmid DNA obtained in this manner by agarose gel electrophoresis and staining with ethidium bromide. DNA from the plasmid pTET3 and DNA from the plasmid pCRY4 may then be isolated from the agarose gel. To this end, the agarose gel containing the plasmid DNA is combined with a chaotropic reagent, the plasmid DNA present in the resultant solution is bound onto the surface of glass or silica gel particles and then eluted back out from this matrix. The person skilled in the art will find detailed instructions relating to this process in the manual xe2x80x9cQIAEX II Handbook for DNA Extraction from Agarose Gelsxe2x80x9d from Qiagen GmbH (Hilden, Germany, 1997). In this manner, it is possible to prepare pTET3 DNA and pCRY4 DNA in pure form.
DNA of the plasmid to be investigated is treated with restriction enzymes individually or in combination as described by Roberts et al. (Nucleic Acids Research 27, 312-313 (1999)). The resultant DNA fragments are separated by agarose gel electrophoresis and the restriction sites assigned. The person skilled in the art will find instructions in this connection, for example, in Rodriguez and Tait xe2x80x9cRecombinant DNA Techniques: An Introductionxe2x80x9d (Addison-Wesley Publishing Company, London, 1983) or in xe2x80x9cGuide to Molecular Cloning Techniquesxe2x80x9d edited by Berger and Kimmel (Methods in Enzymology, Vol. 152, Academic Press, London, 1987). In this manner, the length of the plasmid may be determined or the restriction map plotted. Plasmid pTET3 has a length of approximately 27.8 kbp and is shown in FIG. 1. Plasmid pCRY4 has a length of approximately 48 kbp and is shown in FIG. 2.
Plasmids pTET3 and pCRY4 have a moderate or low copy number. By virtue of this property, they advantageously complement the range of known plasmids for Corynebacterium. Instructions relating to determining copy number may be found, for example, in Miwa et al. (Agricultural and Biological Chemistry 48, 2901-2903 (1984)) and Vohradsky et al. (Electrophoresis 13, 601-612 (1993)).
In order to ensure simple handling of plasmids pTET3 and pCRY4, the DNA region responsible for replication on each plasmid is determined. Known plasmid vectors of Escherichia coli such as for example pK18 (Pridmore, Gene 56, 309-312 (1987)), pK18mob2 (Tauch et al., Plasmid 40, 126-139 (1998)) or pCR2.1 (Invitrogen BV, Groningen, Netherlands), which cannot replicate in coryneform bacteria, but the resistance gene of which is expressed, are used for this purpose. DNA from plasmids pTET3 and pCRY4 is isolated and treated with restriction enzymes. Individual DNA fragments obtained in this manner may optionally in turn be isolated. The DNA of the plasmid vectors used is treated with the same restriction enzymes or such enzymes that produce compatible ends. The resultant DNA molecules are mixed and treated with T4 DNA ligase. These xe2x80x9ccloningxe2x80x9d techniques were known in the prior art and are described in detail in, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)). After transforming a coryneform host, for example Corynebacterium glutamicum, with the ligation mixture and selecting for the resistance gene of the E. coli plasmid vector used, transformants are obtained. Instructions relating to the transformation of coryneform bacteria may be found, for example, in Thierbach et al. (Applied and Environmental Microbiology 29, 356-362 (1988)), in Liebl et al. (FEMS Microbiology Letters 65, 299-304 (1989)) or in Dunican et al. (Bio/Technology 7, 1067-1070 (1989)). The plasmid DNA of these transformants contains DNA segments of pTET3 or pCRY4, which impart the ability to replicate in coryneform bacteria. Examples of these are:
plasmid pTET3-Rep, which consists of the E. coli plasmid pK18mob2 and the replication region of plasmid pTET3 (FIG. 3), and
plasmid pCRY4-Rep, which consists of the E. coli plasmid pK18mob2 and the replication region of plasmid pCRY4 (FIG. 4).
The sections of DNA characterised in this manner are then in turn subcloned into usual vectors suitable for DNA sequencing. Examples of such vectors suitable for DNA sequencing are, for example, the plasmids pGEM-5zf(xe2x88x92) or pGEM-5zf(+) from Promega Corporation (Promega Protocols and Application Guide, Second Edition, 1991, part number Y981, Promega Corporation, Madison, Wis., USA), plasmid pUC19 (Yanish-Perron et al., Gene 33, 103-119 (1985)) or plasmid pK18 (Pridmore, Gene 56, 309-312 (1987)).
DNA sequencing methods are described inter alia in Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America USA, 74, 5463-5467, 1977) and in Zimmermann et al. (Nucleic Acids Research 18, 1067 (1990)).
The resultant DNA sequences may then be investigated using known algorithms or sequence analysis programs, for example the xe2x80x9cSTADEN computer software packagexe2x80x9d (Molecular Biotechnology 5, 233-241 (1996)), Butler""s GCG program (Methods of Biochemical Analysis 39, 74-97 (1998)), Pearson and Lipman""s FASTA algorithm (Proceedings of the National Academy of Sciences USA 85, 2444-2448 (1988)) or Altschul et al.""s BLAST algorithm (Nature Genetics 6, 119-129 (1994)) and compared with the sequence entries available in publicly accessible databases. Publicly accessible nucleotide sequence databases are, for example, the European Molecular Biology Laboratory database (EMBL, Heidelberg, Germany) or the National Center for Biotechnology Information database (NCBI, Bethesda, Md., USA).
The novel DNA sequence responsible for replication of the plasmid pTET3, which sequence is provided by the present invention as SEQ ID NO:1, and which bears the repA gene responsible for replication and the parA gene responsible for stability, was obtained in this manner. The amino acid sequences of the encoded proteins were furthermore deduced from this DNA sequence. SEQ ID NO:2 shows the resultant amino acid sequence of the stabilisation protein ParA, while SEQ ID NO:3 shows the resultant amino acid sequence of the replication protein RepA of pTET3.
The novel DNA sequence responsible for replication of the plasmid pCRY4, which sequence is provided by the present invention as SEQ ID NO:4, and which bears the repA gene responsible for replication of pCRY4, was furthermore obtained in this manner. SEQ ID NO:5 shows the deduced amino acid sequence of the replication protein RepA of plasmid pCRY4.
Few naturally occurring genes that impart resistance to antibiotics in Corynebacterium glutamicum are known. The inventors were accordingly all the more surprised to find that plasmid pTET3 imparts resistance to the antibiotics tetracycline, streptomycin, spectinomycin and sulfamethoxazole.
In order to identify antibiotic resistance genes on new plasmids, the strain to be investigated, in the present case Corynebacterium glutamicum LP-6, and a sensitive control strain, in the present case Corynebacterium glutamicum ATCC13032, are initially tested for resistance or sensitivity to various antibiotics and concentrations of antibiotics. The National Committee of Clinical Laboratory Standards (NCCLS) experimental procedure is preferably used for this purpose (xe2x80x9cMethods for dilution antimicrobial susceptibility tests for bacteria that grow aerobicallyxe2x80x9d, fourth edition; Approved Standard, M7-A4, NCCLS 17(2), (1997)). Using the method of xe2x80x9cApproved Standard M7-A4xe2x80x9d, it is possible to determine inhibition concentrations and thus to ascertain the resistance of the investigated strain of bacteria.
The plasmid to be investigated, in the present case pTET3, is then isolated from strain LP-6 as described above and used to transform a suitable control or indicator strain, in the present case strain ATCC13032. Methods for transforming coryneform bacteria are described, for example, in Thierbach et al. (Applied and Environmental Microbiology 29, 356-362 (1988)), in Liebl et al. (FEMS Microbiology Letters 65, 299-304 (1989)) or in Dunican et al. (Bio/Technology 7, 1067-1070 (1989)). Selection is performed on conventional, complex nutrient media, such as for example brain-heart bouillon or Luria-Bertani medium, which are supplemented with the appropriate antibiotics. The antibiotic and the concentration thereof for this selection process is determined on the basis of the above-mentioned xe2x80x9cApproved Standard, M7-A4xe2x80x9d. In this manner, strain ATCC13032[pTET3], is obtained by selection for tetracycline resistance. The resistance/sensitivity of strain ATCC13032[pTET3) and of the control strain ATCC13032 is then investigated using the above-mentioned method, yielding the result that strain ATCC13032[pTET3] is resistant to the antibiotics tetracycline, streptomycin, spectinomycin and sulfamethoxazole.
This antibiotic resistance was further characterised by cloning and sequencing. To this end, plasmid pTET3 is isolated from strain LP-3 or ATCC13032[pTET3], treated with suitable restriction enzymes, mixed with cloning vectors treated in the same manner and treated with T4 DNA ligase. The ligation mixture is transferred by transformation into a suitable cloning host of Escherichia coli. Selection for transformants is performed on a complex nutrient medium, which is supplemented with the appropriate antibiotic. The person skilled in the art will find instructions relating to this method in Sambrook et al. Examples of suitable cloning vectors are pUC19 (Yanish-Perron et al., Gene 33, 103-119 (1985)), pK18mob2 (Tauch et al., Plasmid 40, 126-139 (1998)) or pCR2.1 (Invitrogen BV, Groningen, Netherlands). Suitable hosts are in particular those E. coli strains with restriction and recombination defects. One example of such a strain is the strain DH5xcex1MCR, which has been described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87, 4645-4649 (1990)). Transformation methods are described, for example, in Hanahan (Journal of Molecular Biology 166, 577-580 (1983)) or Tauch et al. (Plasmid 40, 126-139 (1998)). Transformant selection is performed by using the antibiotics to which plasmid pTET3 imparts resistance. The plasmid DNA of the resultant transformants is then isolated and the cloned DNA fragments of plasmid pTET3 are sequenced. The sequences are then analysed as described above and compared with databases of collected DNA sequences.
The inventors discovered in this manner that the genes which impart resistance to the antibiotics tetracycline, streptomycin, spectinomycin and sulfamethoxazole are located on a continuous DNA fragment. This DNA fragment is shown as a restriction map in FIG. 5. The DNA portion containing the genes tetR, tetA and aadA is shown as a sequence in SEQ ID NO:6 and is provided by the invention.
The amino acid sequences of the protein encoded by the particular gene were furthermore deduced from the ascertained DNA sequence. SEQ ID NO:7 shows the deduced amino acid sequence of the tetracycline resistance protein TetA encoded by the tetA gene and SEQ ID NO:8 shows the deduced amino acid sequence of the spectinomycin/streptomycin resistance protein aadA encoded by the aadA gene. SEQ ID NO:9 shows the coding region of the tetR gene and SEQ ID NO:10 the amino acid sequence of the tetracycline resistance repressor protein TetR.
Coding DNA sequences arising from SEQ ID NO:6 based on the degeneracy of the genetic code are also provided by the present invention. DNA sequences which hybridise with SEQ ID NO:1 or parts of SEQ ID NO:1 are similarly provided by the invention. Conservative substitutions of amino acids in proteins, for example the substitution of glycine for alanine or of aspartic acid for glutamic acid, are known to those of skill in the art as xe2x80x9csense mutationsxe2x80x9d, which result in no fundamental change in activity of the protein, i.e. they are functionally neutral. Amino acid sequences arising in a corresponding manner from SEQ ID NOS:7, 8 and 10 are also provided by the present invention.
The DNA fragments of plasmids pTET3 and pCRY4 from Corynebacterium glutamicum strain LP-6 may then be combined with DNA fragments of known plasmids of other microorganisms, such as for example Escherichia coli or Corynebacterium glutamicum, to yield further, novel plasmid vectors. For the purposes of the present invention, it is preferred to use plasmid DNA from other strains of the species Corynebacterium glutamicum. This approach, known as self cloning, has the advantage that no foreign nucleotide sequences are introduced in the species Corynebacterium glutamicum. Such further developed plasmid vectors may consist solely of constituents of the novel plasmid pTET3, i.e. of a replication region and at least one antibiotic resistance region, which is used as a selection marker. One example of such a vector is the plasmid vector pSELF3-1 shown in FIG. 6. These vectors may, however, also be composed of constituents of a known plasmid and constituents of pTET3 or pCRY4. One example of such a vector is the plasmid vector pSELF1-1 shown in FIG. 7, in which the known cryptic plasmid PGA1 (U.S. Pat. No. 5,175,108) has been provided with the tetA gene which imparts tetracycline resistance of pTET3.
The plasmid vectors constructed from the novel plasmids pTET3 and pCRY4 may advantageously be used for the fermentative production of industrially interesting metabolites such as amino acids, vitamins and nucleotides.
For example, within the framework of the present invention, a lysC(FER) allele of C. glutamicum which encodes a feed-back resistant aspartate kinase was cloned into C. glutamicum ATCC13032 by means of pSELF1-1. In this manner, a self-cloned lysine producing strain of C. glutamicum was produced.
By way of further example, the panD gene coding for aspartate xcex1-decarboxylase from C. glutamicum was cloned into the C. glutamicum strain ATCC13032xcex94ilvA by means of pSELF1-1. In this manner, a self-cloned pantothenic acid producing strain of C. glutamicum was produced.
One very particular advantage of the novel plasmids pTET3 and pCRY4 and further plasmid vectors based thereon is that they exhibit an unusually high level of compatibility with known plasmids or plasmid vectors.
It was thus found that plasmid pTET3 may coexist in the presence of or is compatible with plasmid vectors based on pGA1 (U.S. Pat. No. 5,175,108), pAG3 (U.S. Pat. No. 5,158,891), pBL1 (Santamaria et al., Journal of General Microbiology 130, 2237-2246 (1984)) or on pHM1519 (Miwa et al., Agricultural and Biological Chemistry 48, 2901-2903 (1984)). This compatibility of pTET3 is still retained when the host cell concerned already contains two or more known plasmid vectors, for example a pBL1 derivative and simultaneously a pHM1519 derivative. pTET3""s capacity to coexist with known plasmids or plasmid vectors is ensured over a sufficiently long period of time or for a sufficiently large number of generations.
It has furthermore been found that plasmid pCRY4 may coexist in the simultaneous presence of or is compatible with plasmids pTET3, pGA1 (U.S. Pat. No. 5,175,108) and pGA2 (U.S. Pat. No. 5,175,108) in the presence of plasmid vectors based on pAG3 (U.S. Pat. No. 5,158,891), pBL1 (Santamaria et al., Journal of General Microbiology 130, 2237-2246 (1984)) or on pHM1519 (Miwa et al., Agricultural and Biological Chemistry 48, 2901-2903 (1984)). This compatibility of pCRY3 is still retained when the host cell concerned already contains two or more known plasmid vectors, for example a pBL1 derivative and simultaneously a pHM1519 derivative. pCRY4""s capacity to coexist with known plasmids or plasmid vectors is ensured over a sufficiently long period of time or for a sufficiently large number of generations.
The improved compatibility of plasmids pTET3 and pCRY4 may advantageously be used for improving strains which produce amino acids, vitamins and nucleotides. Sahm and Eggeling (Applied and Environmental Microbiology 65, 1973-1979 (1999)) thus describe the pantothenic acid producing strain ATCC13032xcex94ilvA [pECM3ilvBNCD, pEKEx2panBC]. This strain bears the pHM1519 derivative pECM3ilvBNCD and the pBL1 derivative pEKEx2panBC. It proved possible to achieve a distinct improvement in the performance characteristics of the stated strain, which already contains two plasmids, after transferring the panD gene by means of the plasmid vector pSELF3-1.